Within the Enemy’s Camp: contribution of the granuloma to the dissemination, persistence and transmission of Mycobacterium tuberculosis

Pulmonary tuberculosis, caused by Mycobacterium tuberculosis (M.tb) represents a leading global health concern, with 8.7 million newly emerging cases, and 1.4 million reported deaths annually. Despite an estimated one third of the world’s population being infected, relatively few infected individuals ever develop active clinical disease. The ability of the host to remain latently infected while preventing disease is thought to be due to the generation of a robust type 1 immune response in the lung, capable of controlling, but not clearing, M.tb. A key feature of the type 1 immune response to M.tb is the formation of immune cellular aggregates termed granuloma. The granuloma structure has long been considered a hallmark of host’s protective response toward M.tb. Historically, a correlative relationship between granuloma formation/maintenance and bacterial control has been seen in models where disrupted granuloma formation or structure was found to be fatal. Despite this established relationship much about the granuloma’s role in M.tb immunity remains unknown. Recent publications suggest that the granuloma actually aids the persistence of M.tb and that the development of a necrotic granuloma is essential to person-to-person transmission. Our group and others have recently demonstrated that enclosed within the granuloma is a population of immunologically altered antigen-presenting cells and T lymphocyte populations. Of note, the ability of these populations to produce type 1 cytokines such as interferon-gamma, and bactericidal products including nitric oxide, are significantly reduced, while remaining competent to produce high levels immunosuppressive interleukin-10. These observations indicate that although the chronic granuloma represents a highly unique environment, it is more similar to that of a tumor than an active site of bacterial control. In this review we will explore what is known about this unique environment and its contribution to the persistence of M.tb

Regulation of TB vaccine-induced airway luminal T cells by respiratory exposure to endotoxin.

Tuberculosis (TB) vaccine-induced airway luminal T cells (ALT) have recently been shown to be critical to host defense against pulmonary TB. However, the mechanisms that maintain memory ALT remain poorly understood. In particular, whether respiratory mucosal exposure to environmental agents such as endotoxin may regulate the size of vaccine-induced ALT population is still unclear. Using a murine model of respiratory genetic TB vaccination and respiratory LPS exposure, we have addressed this issue in the current study. We have found that single or repeated LPS exposure increases the number of antigen-specific ALT which are capable of robust secondary responses to pulmonary mycobacterial challenge. To investigate the potential mechanisms by which LPS exposure modulates the ALT population, we have examined the role of ALT proliferation and peripheral T cell recruitment. We have found that LPS exposure-increased ALT is not dependent on increased ALT proliferation as respiratory LPS exposure does not significantly increase the rate of proliferation of ALT. But rather, we find it to be dependent upon the recruitment of peripheral T cells into the airway lumen as blockade of peripheral T cell supplies markedly reduces the initially increased ALT. Thus, our data suggest that environmental exposure to airborne agents such as endotoxin has a profound modulatory effect on TB vaccine-elicited T cells within the respiratory tract. Our study provides a new, M.tb antigen-independent mechanism by which the respiratory mucosal anti-TB memory T cells may be maintained

Respiratory LPS exposure-increased airway luminal T cells in the lung of vaccinated animals is dependent on the recruitment of peripheral T cells.

<p>(A) Experimental schema; At 1 day after each FTY720 delivery, the deficiency of peripheral blood T cells was verified. The representative histogram is shown (B). At 5 days post-last FTY720 or -vehicle delivery, mice were sacrificed and BAL and lung cells were isolated, and Ag85A tetramer (Tet)-specific (C) and IFN-γ-secreting (D) CD8⁺ T cells in BAL or lung interstitium were determined by immunostaining, intracellular cytokine staining and FACS. Representative dotplots are shown with the average frequency ± SEM from three animals/group. The data in graphs are expressed as mean value ± SEM of three animals per group, representative of two independent experiments.</p

Repeated respiratory LPS exposure further increases vaccine-induced T cells in airway lumen (BAL) and lung interstitium at 14 days post-last LPS delivery.

<p>(A) Experimental schema; At 14 days post-last LPS or –PBS delivery, mice were sacrificed and BAL and lung cells were isolated, and Ag85A tetramer (Tet)-specific (B) and IFN-γ-secreting (C) CD8⁺ T cells in BAL or lung interstitium were determined by immunostaining, intracellular cytokine staining and FACS. Representative dotplots are shown with the average frequency ± SEM from three animals/group. Data in graphs are expressed as mean value ± SEM of three animals per group.</p

Respiratory LPS exposure-increased airway luminal T cells are able to undergo enhanced secondary T cell responses to pulmonary mycobacterial challenge.

<p>(A) Experimental schema. At 7 days post-mycobacterial challenge, mice were sacrificed and BAL and lung cells were isolated, and Ag85A tetramer (Tet)-specific (B) and IFN-γ-secreting (C) CD8⁺ T cells in BAL or lung interstitium were determined by immunostaining, intracellular cytokine staining and FACS. Representative dotplots are shown with the average frequency ± SEM from three animals/group. Data in graphs are expressed as mean value ± SEM of three animals per group.</p

Increased levels of proinflammatory cytokines/chemokines in the airway lumen of LPS-exposed and vaccinated animals.

<p>BAL fluids were collected from the lung of the animals set up according to Fig. 1A experimental schema. Cytokine and chemokine contents were determined by Luminex or ELISA assays. Data were expressed as mean value ± SEM of three animals per group.</p

Respiratory LPS exposure increases vaccine-induced T cells in airway lumen (BAL) and lung interstitium at 14 days.

<p>(A) Experimental schema; At 14 days post-LPS or -PBS, mice were sacrificed and BAL and lung cells were isolated, and Ag85A tetramer (Tet)-specific (B) and IFN-γ-secreting (C) CD8⁺ T cells in BAL or lung interstitium were determined by immunostaining, intracellular cytokine staining and FACS. Representative dotplots are shown with the average frequency ± SEM from three animals/group. The data in graphs are expressed as mean value ± SEM of three animals per group, representative of three independent experiments.</p

Respiratory LPS exposure increases vaccine-induced T cells in airway lumen (BAL) and lung interstitium at 7 days.

<p>(A) Experimental schema; At 7 days post-LPS or -PBS, mice were sacrificed and BAL and lung cells were isolated, and Ag85A tetramer (Tet)-specific (B) and IFN-γ-secreting (C) CD8⁺ T cells in BAL or lung interstitium were determined by immunostaining, intracellular cytokine staining and FACS. Representative dotplots are shown with the average frequency ± SEM from three animals/group. The data in graphs are expressed as mean value ± SEM of three animals per group.</p

Continuous and Discontinuous Cigarette Smoke Exposure Differentially Affects Protective Th1 Immunity against Pulmonary Tuberculosis

<div><p>Pulmonary tuberculosis (TB), caused by <i>Mycobacterium tuberculosis,</i> is the leading cause of death due to a bacterial pathogen. Emerging epidemiologic evidence suggests that the leading risk factor associated with TB mortality is cigarette smoke exposure. Despite this, it remains poorly understood what is the effect of cigarette smoke exposure on anti-TB immunity and whether its potential detrimental effect can be reversed by cigarette smoking cessation. In our current study, we have investigated the impact of both continuous and discontinuous cigarette smoke exposure on the development of anti-mycobacterial type 1 immunity in murine models. We find that while continuous cigarette smoke exposure severely impairs type 1 immunity in the lung, a short-term smoking cessation allows rapid restoration of anti-mycobacterial immunity. The ability of continuous cigarette smoke exposure to dampen type 1 protective immunity is attributed locally to its affects on innate immune cells in the lung. Continuous cigarette smoke exposure locally, by not systemically, impairs APC accumulation and their production of TNF, IL-12, and RANTES, blunts the recruitment of CD4+IFN-γ+ T cells to the lung, and weakens the formation of granuloma. On the other hand, smoking cessation was found to help restore type 1 immunity by rapidly improving the functionality of lung APCs, enhancing the recruitment of CD4+IFN-γ+ T cells to the lung, and promoting the formation of granuloma. Our study for the first time demonstrates that continuous, but not discontinuous, cigarette smoke exposure severely impedes the lung expression of anti-TB Th1 immunity via inhibiting innate immune activation and lung T cell recruitment. Our findings thus suggest cigarette smoking cessation to be beneficial to the control of pulmonary TB.</p> </div

Continuous, but not discontinuous smoke exposure, impairs the production of type 1 cytokines while enhancing the production of IL-4, and reducing the production of bactericidal nitric oxide by lung MNCs following mycobacterial infection.

<p>Following the exposure-challenge model described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059185#pone-0059185-g002" target="_blank">Figure 2A</a>, we evaluated the impact of cigarette smoke exposure on the production of type 1 &2 cytokines and nitric oxide by mycobacteria infected lung MNCs. Following 48 hr lung MNC culture, the levels of TNF (A), IL-12p40 (B), IFN-γ (C), IL-4 (D) were evaluated by cytokine ELISA, and production of nitric oxide (E) by a modified Griess assay. Values represent the mean and standard error for 5 mice per exposure protocol. *p≤0.05; **p≤0.01; ***p≤0.001.</p